1. Field of the Invention
The present invention relates generally to the fields of molecular biology and enzymology. More particularly, it concerns the isolation and characterization of human palmitoyltransferase complexes, and uses therefor.
2. Description of Related Art
Dozens of cellular and viral proteins are post-translationally modified with palmitate or other long-chain fatty acids through a reversible thioester linkage (Linder & Deschenes, 2003; Linder, 2001; Schlesinger et al., 1993). Examples include cell surface receptors, viral glycoproteins, and signal transducers including Ras, heterotrimeric G proteins, and non-receptor tyrosine kinases. Palmitoylation is almost exclusively a property of membrane proteins and can occur on intracellular membranes early in the secretory pathway, or later at the plasma membrane. Although this modification was first described over 30 years ago, the molecular mechanism of palmitate addition has yet to be elucidated and continues to be a matter of some controversy.
A variety of approaches have been used in an attempt to identify the enzyme(s) responsible for this function. A palmitoyltransferase activity assayed using mammalian H-Ras as a substrate was purified and identified as thiolase A, an enzyme required for fatty acid-oxidation (Liu et al., 1996a; Liu et al., 1996b). The localization of this enzyme in peroxisomes makes it an unlikely candidate for a physiological regulator of Ras palmitoylation. Palmitoyltransferase activities assayed using viral glycoproteins, the non-receptor tyrosine kinase p59fyn, or G-protein heterotrimer as substrates have been detergent-solubilized, but the instability of the activity has hampered purification and molecular identification (Dunphy et al., 1996, Dunphy et al., 2001; Berthiaume and Resh, 1995; Schmidt and Burns, 1989).
Accordingly, the identification of bona fide palmitoyl acyl transferases and the palmitoyl acyl transferase for Ras oncogene proteins remains an unachieved goal. In the absence of such an identification, the ability to screen for drugs that modulate the function of this family of enzymes remains limited.